hms at shapirolab.com
Tue Jun 6 15:30:08 EDT 2006
Elaine Kunze wrote:
>We want to ennumerate the number of live bacteria in a gel. The
>bugs are Staph epidermidis. We are using the BacLite kit. We see a
>number of green only bacteria under the microscope despite the fact
>that nothing cultures after three weeks. We do have some PI reds,
>but there are many green onlys. What am I missing?
Dye exclusion is not the best indicator of nonviability in bacteria
(or, for that matter, in mammalian cells). The fact that bacteria
stain green with the BacLite kit only indicates that their membranes
are intact, not that they are either metabolically or reproductively
viable. In fact, there is evidence that some viable bacteria will,
under certain circumstances, take up PI.
You might try 30 uM DiOC2(3), which (provided most of it does not
stick to the gel material) will be concentrated enough in
metabolically viable bacteria, i.e., those with membrane potentials,
to give red-orange (>600 nm) fluorescence excitable with green light
- no guarantees on this in microscopy, although the red/green
fluorescence ratio is pretty good for measuring membrane potential in flow.
Since some of us are now trying trying to make it easier to identify
postings to this list that are relevant to microbial cytometry, it
would be helpful if anyone making additional postings to this thread
or on the subject added the keyword [Microbial] to the title, as I
have done. Thanks.
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